There are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response [Schroff, R. W. et al (1985) Cancer Res. 45: 879-885; Shawler, D. L. et al (1985) J. Immunol. 135: 1530-1535]. For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response [WO 89/09622; EP 0239400; EP 0438310; WO 91/06667]. These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant “humanised” antibodies have, in several cases, still elicited an immune response in patients [Issacs J. D. (1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999) Transplantation 68: 1417-1420].
Antibodies are not the only class of polypeptide molecule administered as a therapeutic agent against which an immune response may be mounted. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans. Notable examples include the therapeutic use of granulocyte-macrophage colony stimulating factor [Wadhwa, M. et al (1999) Clin. Cancer Res. 5: 1353-1361] and interferon alpha 2 [Russo, D. et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl. J. Med. 318: 1409-1413] amongst others.
An immune response to a therapeutic protein proceeds via the MHC class II peptide presentation pathway. Here exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP type. MHC Class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others. Engagement of a MHC class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
A principal factor in the induction of an immune response therefore is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC class II molecules, so-called “T-cell epitopes”. Such potential T-cell epitopes are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Such T-cell epitopes can be measured to establish MHC binding. Implicitly, a “T-cell epitope” means an epitope which when bound to MHC molecules can be recognized by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response. It is, however, usually understood that certain peptides which are found to bind to MHC Class II molecules may be retained in a protein sequence because such peptides are recognised as “self” within the organism into which the final protein is administered.
From the forgoing, it is clear that immunogenicity of a therapeutic protein strongly depends on the ability of the immune system to select and proliferate T-cell clones that are specific for peptides derived from the therapeutic protein. The activation of a specific T-cell clone is a complex event but one that occurs at the end of a pathway of complex events. The pathway can be characterised by several key steps;                1) protein uptake by antigen presenting cells;        2) proteolytic processing of the protein by proteases in the antigen presenting cells;        3) binding of peptides excised from the protein to the MHC molecules that are able to present these on the cell surface;        4) transport of peptides MHC complexes to the cell surface.        
The present invention is concerned with solutions to the problem posed by the inevitable presence of immunogenic MHC class II epitopes within therapeutic proteins. In general aspect, the invention is concerned with disrupting the ability of peptide sequences to emerge from the above outlined pathway, and in particular provides methods by which steps 2-4, as outlined above, may be manipulated in the development of a therapeutic protein with an improved immunogenic profile.
In the art there are procedures for identifying synthetic peptides able to bind MHC class II molecules. Such peptides may not function as T-cell epitopes in all situations, particularly, in vivo due to the processing pathways or other phenomena. T-cell epitope identification may be considered as the first step to epitope elimination and computational techniques such as scanning for recognised sequence motifs in experimentally determined T-cell epitopes or by using computational techniques to predict MHC class II-binding peptides have been published. WO98/52976 and WO00/34317 teach computational threading approaches to identifying polypeptide sequences with the potential to bind a sub-set of human MHC class II DR allotypes. In these teachings, predicted T-cell epitopes are removed by the use of judicious amino acid substitution within the primary sequence of the therapeutic antibody or non-antibody protein of both non-human and human derivation. These procedures provide modified polypeptide sequences by amino acid substitution and do not anticipate the use of other modifying modalities in order to eliminate the epitope.
Other techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides and able to bind to T-cell clones from peripheral blood samples from human or experimental animal subjects have been used in the art [Kern, F. et al (1998) Nature Medicine 4:975-978; Kwok, W. W. et al (2001) TRENDS in Immunol. 22:583-588] and may equally be exploited in an epitope identification strategy, but also do not provide means for epitope elimination.
U.S. Pat. No. 5,833,991 (Masucci) provides a method for preventing undesired immune responses to recombinant proteins exploiting tracts of glycine-containing sequence, this approach is similar to widely practiced methods whereby an immunologically inhert species is adducted to a therapeutic protein for example a polymeric molecule such as PEG and has been intensively described in the art [for example schemes see U.S. Pat. No. 5,349,001 and WO90/13590].
For clarity in conveying understanding to the present invention, the major components of what we herein term the “immune processing pathway” are now described.
MHC Class II System
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins, however, isotypes HLA-DQ and HLA-DP perform similar functions and are biologically relevant. The MHC class II DR molecule is made of an alpha and a beta chain that insert at their C-termini through the cell membrane. Each hetero-dimer possesses a ligand binding domain which binds to peptides varying between 9 and 20 amino acids in length, although the binding groove can accommodate a maximum of 11 amino acids. The ligand binding domain is comprised of amino acids 1 to 85 of the alpha chain, and amino acids 1 to 94 of the beta chain. DQ molecules have recently been shown to have an homologous structure and the DP family proteins are also expected to be very similar. In humans approximately 70 different allotypes of the DR isotype are known, for DQ there are 30 different allotypes and for DP 47 different allotypes are known. Each individual bears two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and such structures point to an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide [Brown et al Nature (1993) 364: 33; Stern et al (1994) Nature 368: 215]. Polymorphism identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
Proteolytic Processing
Protein antigens can be taken up by various mammalian cells for processing and APCs expressing MHC class II molecules are able to do this with particular efficiency. Antigens can enter the endocytic route by various mechanisms, such as receptor-mediated endocytosis, phagocytosis, macropinocytosis and autophagy. The antigen is degraded in endocytic vesicles, which are acidic and proteolyticaly active. Multiple different proteases participate in this pathway and many of which have not yet been characterised. Endocytic vesicles gradually change in character, becoming more acidic and more proteolyticaly active. The antigen is degraded in steps and the most sensitive or exposed areas will be attacked first.
Many different proteases have been identified in the endocytic vesicles of antigen presenting cells. Since most of these proteins are also found in other proteolytic processes in mammals, some are well characterised. In B cells and dendritic cells cathepsin S plays a key role, in thymus endothelial cells this is cathepsin L and in macrophages cathepsin F.
Cathepsins are papain family cysteine proteases involved in a variety of physiologic processes in addition to antigen presentation. Cathepsins are glycoproteins and contain an essential cysteine residue in their active site but differ in some enzymatic properties, including substrate specificities and pH stability. Some cathepsins have been identified have ubiquitous expression and may have ‘housekeeping’ roles, whereas others, like cathepsin S, have tissue-restricted expression and may have more specific functions.
In addition to cathepsins S, L and F, several other cathepsins involved in antigen degradation have been identified. These are cathepsin B, cathepsin H, cathepsin D and cathepsin E and potentially cathepsins Z, V and K. Since the pH-optimum for the various cathepsins differs, some are likely to be more active in early endosomes, while others play a role in the later stages of antigen processing. As a consequence the population of protein fragments will vary through the processing pathway, while there may also be a different range of peptides in different individual APCs. In addition to the cathepsins, other proteases such as asparagine endopeptidase, which plays a crucial role in the processing of antigens taken up by B-cells, may also be involved in the degradation of antigens.
Formation of the MHC Peptide Complex
Following the antigen degradation pathway, peptides will emerge that have the potential to bind in the binding groove of HLA-DR, HLA-DQ or HLA-DP molecules. In order for a peptide to bind to HLA-DR, HLA-DQ or HLA-DP molecules, it has to remove a peptide called CLIP (class II associated Ii peptide) from the binding groove. CLIP is a peptide derived from the Ii protein, which is a chaperone molecule, targeting HLA-DR, DQ and DP from the endoplasmic reticulum to the endocytic vesicles. Since Ii contains a (C-terminal) trimerization domain, nonameric complexes are formed. The cytoplasmic domain of Ii contains signal sequences, which target the complexes through the Golgi into the endocytic route. Here the Ii molecules are degraded in several stages. First the trimerization domain is cleaved off by a non-cysteine protease, so the nonamer dissociates leaving DRα-DRβ-IiP22 complexes. A Cys-protease cleaves C-terminally of the CLIP-region, thereby removing two bulky carbohydrates, leaving DRα-DRβ-IiP10. Finally Ii is cleaved at the N-terminus of the CLIP peptide, leaving DRα-DRβ-CLIP. The CLIP peptide can be exchanged for other peptides that are present in the endosomes.
In the exchange reaction of peptides binding to HLA-DR a molecule named HLA-DM, a MHC class Ii encoded enzyme, plays a key role by catalysing the exchange of CLIP peptides and other peptides that are bound to DRα-DRβ with a low affinity, for more stably binding peptides derived from processed antigens [reviewed by Busch et al., (2000), Curr. Op. Immunol. 12, 99-106]. HLA-DM has a great effect on the kinetics of the exchange reaction of peptides to purified HLA-DR in vitro, efficiently stimulating the release of certain peptides. The stoichiometry of HLA-DM and HLA-DR in endosomes is 1:5-1:12, while the turnover of DM in vitro is ca. 3-12/min. HLA-DM interacts with HLA-DR molecules via exposed hydrophobic regions and charged residues. The most likely sites of interaction have been proposed on a crystal structure model of HLA-DM [Mosyak et al., (1998) Immunity 9:377-383]. HLA-DM preferentially binds to HLA-DR complexes to which a peptide has bound with low affinity. Besides this, it can also bind to empty HLA-DR dimers, which are unstable and likely to aggregate in the absence of HLA-DM. The binding of HLA-DM stabilises the ‘empty state’ of the HLA-DR dimers to which peptide is bound loosely or no peptide is bound at all. By keeping the binding groove of HLA-DR open in this way, peptides can compete for binding in this groove. The exchange of peptides can also take place in the absence of HLA-DM in vivo, albeit with a significantly reduced efficiency. The N-terminal domain of the CLIP peptide can interact with some HLA-DR allotypes outside the binding groove and thereby stabilise a conformation in which the CLIP peptide is more likely to be released. HLA-DM action is also no absolute requirement for the transport of HLA-DR complexes to the plasma membrane. In the absence of HLA-DM, class II molecules with CLIP peptides of self-peptides still bound can make their way to the cell surface.
HLA-DM has maximal activity at acidic pH and will therefore be mainly active in the proteolytic endosomes (called MIIC). In the acidic MIICs HLA-DM will be discharged after a peptide has stably bound in the antigen binding groove of HLA-DR. Alternatively HLA-DM can be co-transported with the HLA-DR complex to the cell surface, where the neutral pH leads to a quick release. Indeed, small amounts of HLA-DM can be found at the cell surface, where they may have a functional role [Arndt et al. (2000), EMBO J. 19:6, 1241-1251]. Subsequently HLA-DM, which contains a lysosomal targeting signal, is quickly internalised and retargeted to the MIICs.
An MHC class II encoded protein named HLA-DO has a regulatory function [reviewed by van Ham et al., (2000), Immunogen. 51, 765-770], inhibiting HLA-DM activity in a pH dependent manner [van Ham et al. (1997), Curr. Biol. 7, 950-957]. HLA-DM has optimal activity at pH 5, but is also active at pH 6. Binding of HLA-DO at pH6 abolishes HLA-DM activity. Thus, HLA-DO acts as a pH sensor for HLA-DM activity, inhibiting it in early endosomes but allowing activity at lysosomal pH. Indeed in HLA-DO minus cells, HLA-DR can be found loaded with long peptides that have not yet been fully processed, while in HLA-DO positive cells binding of those peptides to HLA-DR does not take place.
While HLA-DM is expressed in all APCs, HLA-DO is mainly found in B-cells. It has been suggested that this is a way to specifically stimulate the presentation of epitopes derived from antigens that were internalised through B-cell receptor mediated uptake. When antibody-bound antigens are endocytosed by B-cells, they are quickly transported to MIIC, the late-stage protein processing vesicles. Since HLA-DO is not functioning in these compartments because of the acidic pH, peptides that are excised from these antigens will quickly bind to HLA-DR. Peptides produced in early endosomes, i.e. endosomes where HLA-DO suppresses HLA-DM function due to the pH, from antigens taken up by non-receptor mediated endocytosis, will be prevented from binding to HLA-DR [van Ham et al. (2000), J. Exp. Med 191:7, 1127-1136].
The binding groove of HLA-DR, DQ and DP dimers contain several pockets in which amino acids of the antigenic peptide may bind. The so-called anchor residues of peptides, which may bind in these pockets, are the main determinants for binding of peptides to HLA-DR, DQ and DP. Binding to the MHC is a competitive process and peptides with high affinity are known to compete successfully for lower affinity peptides favouring their presentation on the surface of the APC [Adorni L. et al (1988) J. Exp. Med 168:2091; Ii. W. et al (1992) Eur. J. Immunol. 22: 943]. For some peptides the affinity is so high as to constitute effectively an irreversible binding reaction [Lanzavecchia A. et al (1992) Nature 357: 249].